Two-hybrid method

McCormack D A, Kroes G J and Neuhauser D 1998 Resonance affected scattering Comparison of two hybrid methods involving filter diagonalization and the Lanczos method J. Chem. Phys. 109 5177... 

Brent, R., and Finley, R. L., Jr. (1997). Understanding gene and allele function with two-hybrid methods. Annu. Rev. Genet. 31, 663-704. 

Resonance Affected Scattering Comparison of Two Hybrid Methods Involving Filter Diagonalization and the Lanczos Method. 

Many variants of the two-hybrid system have been devised.d For example, a green fluorescent protein reporter can be used/ Because significant biological protein-protein interactions often require that three or more proteins interact,d hybrid systems involving more than two proteins have been developed. Two-hybrid systems for bacteria have also been devised.1" A virtue of the two-hybrid methods is that they work with undenatured, if not totally natural, proteins. This is in contrast to widely used methods that involve separation of denatured proteins on gels or columns. 

Interpretation of results of these studies is still difficult. Results of two-hybrid methods become more useful if they can be coordinated with other approaches. For example, computational methods can predict interactions from genome sequences alone. 11/0 More than 45,000 interactions have been predicted among yeast proteins. Reliable identification of such motifs as DNA-binding domains and Ca2+- binding domains can complement two-hybrid analysis.11 The yeast genome is predicted to contain 162 coiled-coil sequences and at least 213 unique interactions between them.0 Examination of sequences of protein families in the Protein Data Bank (PDB) led to prediction of 8151 interactions of 664 types between protein families in yeast.P... 

Whether we discuss silk, proteins embedded in membranes, or soluble complexes of cytosolic proteins, we must ask questions about interactions. A first step is to identify interactions720-730 among proteins either in vitro or in living cells.731 Proteomic methods, which include the yeast two-hybrid method (Box 29-F), are widely used for this purpose. It is possible to identify large sets of interacting proteins, to identify disease states, to observe effects of drugs, and to compare metabolism among species. 

The DFT methods are all well behaved, and perform surprisingly well for such a difficult system. The two hybrid methods, B3LYP and B3PW91, give results comparable to tlrose for CCSD(T). The main problem is of course that there is no way of... 

Figure 1 The classic yeast two-hybrid method and derivatives, (a) Schematic diagram of the yeast two-hybrid approach, describing an interaction between protein X and protein Y. Protein X is fused to a transcription factor DNA-binding domain (the "bait" construct), and protein Y is fused to a transcription factor activation domain (the "prey" construct), (b) High-throughput applications of the yeast two-hybrid method use mating of haploid strains carrying bait and prey, respectively. Hybrids can be mated in arrayed formats (as shown) or as libraries, (c) The reverse two-hybrid method uses a counter-selectable marker to indicate loss of protein interaction because of disruption by an inhibitor protein/small molecule ("/" illustrated in the diagram) or mutation(s) in proteins X and/or Y.

We see two major appearing frontiers for new kinds of molecular data. The first is proteomics (See Chapter 4 of volume 2) and metabolomics. With a combination of 2D gel, mass spectrometry, protein microarray and yeast-two-hybrid methods, a large amount of protein sequence, expression, and interaction data will be produced on a cell-wide level. On the one hand, bioinformatics has to address the challenge of interpreting these data. On the other hand, especially the protein interaction data will provide an interesting basis for probing deeper into the details of regulatory networks. Such data are collected in special protein interaction databases such as DIP [9,10] and BIND [11],... 

While the two-hybrid method has been extensively used to detect natural protein-protein interactions, it should also be well suited for protein evolution. Brent and coworkers demonstrated that the two-hybrid assay can be used to... 

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